MS is an autoimmune disease potentially mediated by activated myelin reactive Th1/Th17 T cells in a genetically susceptible host. Kuchroo recently discovered that Th1 cells selectively express TIM-3, and its ligation regulates tolerance induction. New, complementary data sets generated in the Hafler &Kuchroo labs demonstrate that TIM-3 is also expressed on APCs, including both dendritic cells and monocytes. Contrary to the consequences of TIM-3 engagement on T cells, ligation of TIM-3 on APCs may promote Th1-mediated immunity in that stimulation of monocytes with the TIM-3 ligand galectin-9 (Gal-9) induces TNF-a and IL-6 secretion. Moreover, we have demonstrated that microglia in CNS white matter express high levels of TIM-3, and glial expression of TIM-3 and Gal-9 are up-regulated in MS lesions. This leads us to hypothesize that the altered expression or function of TIM-3 by T cells and circulating APCs and CNS microglia may alter the balance of Th1 differentiation, and perhaps is associated with chronic CNS inflammation due to up-regulation of Gal-9 expression on astrocytes, inducing the activation of TIM-3* microglia. Thus, it is possible that the innate TIM-3/Gal-9 pathway may be involved with transition to secondary progressive disease in patients with MS. Experiments in Aim 1 have been designed to provide us with a detailed mechanistic understanding of how TIM-3 expression on APCs modulates their activity, and how this may subsequently influence the balance of effector versus regulatory T cell differentiation. In Aim 1b, based on our discovery of the presence of TIM-3 on CD11b+ microglia in white matter of normal brain, and the coordinated modulation of TIM-3 &Gal-9 expression depending on the nature of CNS inflammation, we will determine how this pathway modulates glial responses within the CNS. In collaboration with Kuchroo, we propose experiments to examine the kinetics of TIM-3 induction on microglia associated with migration of hematopoietic cells into the CNS. Aim 2 will investigate our recent discovery that 5 distinct TIM- 3 splice variants exist in humans, including membrane &soluble forms with variable potential signaling capacity. Using quantitative RT-PCR analysis in conjunction with techniques to modulate specific TIM-3 isoforms, we will explore how relative differences in the expression of these TIM-3 isoforms may influence T cell vs. APC biology. Our third aim will extend our analysis of TIM-3 regulation of APC function by defining the TIM-3 signaling pathways in T cell vs. monocyte lineage cells. We will perform structure/function experiments to determine which structural elements in the cytoplasmic tail of TIM-3 are responsible for its effects on cytokine production and downstream signaling pathways in TIM-3+ T cells and monocytic cells, and will determine which cytoplasmic signaling proteins are recruited to the cytoplasmic tail of TIM-3 to mediate activation of downstream signaling pathways. These experiments will determine how differences in TIM-3 isoform expression and signaling dictate divergent functional outcomes in adaptive versus innate immunity and how this innate pathway may be involved in the pathoaenesis of autoimmune disease.